BARD1 Gene Polymorphisms Confer Nephroblastoma Susceptibility

The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger-deficient variant BARD1beta. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1beta coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1beta. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1beta led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1beta in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1beta.

<div>Abstract<p>The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger–deficient variant BARD1β. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1β coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1β. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1β led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1β in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1β. [Cancer Res 2009;69(3):1125–34]</p></div>

<div>Abstract<p>The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger–deficient variant BARD1β. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1β coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1β. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1β led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1β in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1β. [Cancer Res 2009;69(3):1125–34]</p></div>

Background & Aims: BRCA1 associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex. Its alterations impact function of BRCA1, disabling HR (homologous recombination) repair leading to sensitivity to PARP-1 inhibition in cancer. In colorectal cancer (CRC), we observed expression of BARD1 splice variants (SVs) and loss of full-length is associated with poor prognosis despite wild type expression of BRCA1. Here, we examined the roles of BARD1 SV and its therapeutic impact on PARP-1 inhibition, oxaliplatin and irinotecan sensitivity in CRC cell lines. Methods: A panel of CRC cell lines was treated with PARP-1 inhibitor, ABT888, and HR proficiency assessed via RAD51 foci formation. BARD1 splice variant (SV B1/4) transcription was determined via real-time PCR, and protein expression assessed via immunoblotting. Tumorigenesis of CRC cells expressing SV B1/4 and wild type BARD1 was measured by epithelial-mesenchymal transitions (EMT), colony formation abilities, and migration assays. Results: Caco-2 CRC cells were significantly more sensitive to PARP-1 inhibition when compared to the other CRC cell lines. This sensitivity was not associated with loss of BRCA1 or full-length BARD1 expression but rather with expression of BARD1 SV 1/4. Caco-2 showed significantly impaired HR response following PARP-1 inhibition with ABT888 and characterized by decreased RAD51 foci formation. Exogenous expression of the BARD1 SV B1/4 in SW480 cells exhibits a more transformed phenotype, decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and increased sensitivity to PARP-1 inhibitor. Addition of irinotecan to ABT888 pre-treated cells created higher specificity and sensitivity with respect to killing of SV B1/4 expressing SW480 cells. Further immunofluorescence and confocal microscopy studies are underway to investigate the effect of BARD1 SV B1/4 on BRCA1 homologous recombination DNA repair function. Conclusions: Expression of BARD1 SV might be a potential biomarker for therapeutic sensitivity to PARP-1 inhibitors in treatment of CRC. Citation Format: Ozkan Ozden, Faraz Bishehsari, Jessica Bauer, Seung Hyua Baik, Barbara Jung. Expression of BARD1 splice variant impairs homologous recombination and can predict PARP1 inhibitor therapy in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3626. doi:10.1158/1538-7445.AM2015-3626

The breast cancer susceptibility protein BRCA1 and its partner BRCA1-associated RING domain protein 1 (BARD1) form an E3-ubiquitin (Ub) ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1–BARD1 in postmitotic cells, such as neurons, remain poorly defined. Here, we report that BRC-1 and BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gqα–diacylglycerol (DAG) signaling pathway. This pathway is downregulated by DAG kinase (DGK), which converts DAG to phosphatidic acid (PA). We demonstrate that inactivation of DGK-3 suppresses the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1–BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1–BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1–BRD-1 complex regulates axon regeneration in concert with the Gqα–DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.SIGNIFICANCE STATEMENT BRCA1–BRCA1-associated RING domain protein 1 (BARD1) is an E3-ubiquitin (Ub) ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1–BARD1 in postmitotic cells, such as neurons, remain poorly defined. We show here that Caenorhabditis elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase (DGK)-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1–BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1–BARD1 in fully-differentiated neurons.

Thermodynamic study of the BRCT domain of BARD1 and its interaction with the -pSER-X-X-Phe- motif-containing BRIP1 peptide

The fellowship of the RING: BRCA1, its partner BARD1 and their liaison in DNA repair and cancer

Current oncological developments are based on improved understanding of genetics, and especially the discovery of genes whose alterations affect cell functions with consequences for the whole body. Our work is focused on the one of these genes, BRCA1-associated RING domain protein 1 (BARD1), and its oncogenic role in breast cancer. Most importantly, the study points to new avenues in the treatment and prevention of the most frequent female cancer based on BARD1 research. The BARD1 and BRCA1 (BReast CAncer type 1) proteins have similar structures and functions, and they combine to form the new molecule BARD1-BRCA1 heterodimer. The BARD1-BRCA1 complex is involved in genetic stabilization at the cellular level. It allows to mark abnormal DNA fragments by attaching ubiquitin to them. In addition, it blocks (by ubiquitination of RNA polymerase II) the transcription of damaged DNA. Ubiquitination, as well as stabilizing chromatin, or regulating the number of centrosomes, confirms the protective cooperation of BARD1 and BRCA1 in the stabilization of the genome. The overexpression of the oncogenic isoforms BARD1β and BARD1δ permit cancer development. The introduction of routine tests, for instance, to identify the presence of the BARD1β isoform, would make it possible to detect patients at high risk of developing cancer. On the other hand, introducing BARD1δ isoform blocking therapy, which would reduce estrogen sensitivity, may be a new line of cancer therapy with potential to modulate responses to existing treatments. It is possible that the BARD 1 gene offers new hope for improving breast cancer therapy.

BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1β may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer.

BRCA1-associated RING domain protein 1 (BARD1) gene polymorphisms may be associated with neuroblastoma (NB) susceptibility. However, the results remain controversial. Relevant studies were identified by searching PubMed, Web of Science, Embase, China National Knowledge Infrastructure databases up to March 5, 2023. The strength of the association between BARD1 polymorphisms and susceptibility of NB was assessed by calculating odds ratios (ORs) and 95% confidence intervals (95% CIs) through the fixed- or random-effects model. Eight articles involving 12 studies were finally included. We found that rs6435862 T > G, rs3768716 A > G, rs17487792 C > T and rs7587476 C > T variant increase the risk of NB in allelic, dominant, recessive, homozygous and heterozygous genetic models, while rs7585356 G > A variant appeared protective against NB. When stratified by ethnicity, subgroup analysis indicated that the above association remained significant in Caucasian populations in all genetic models, except for rs7585356G > A polymorphism in Asians. In Asian populations, we found the similar results in the allelic and dominant model of rs6435862 T > G, rs3768716 A > G, rs17487792 C > T and rs7587476 C > T as in Caucasians, while there lacked a significant association in the other three model. In addition, rs7585356 G > A was not associated with an increased risk of NB in the Asian population. After Bonferroni correction, significant associations for rs7585356 G > A disappeared in both Asian and Caucasian populations, with no significant association found for rs7587476 in the allelic and dominant models among Asians. BARD1 polymorphisms might be significantly associated with NB susceptibility. It is crucial that these finding should be further confirmed through extensive and well-planned studies.

Computational structural assessment of BReast CAncer type 1 susceptibility protein (BRCA1) and BRCA1-Associated Ring Domain protein 1 (BARD1) mutations on the protein-protein interface.

Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.

Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.

BRCA1 associated ring domain protein 1(BARD1) is a tumor suppressor protein having a wide role in cellular processes like cell-cycle checkpoint, DNA damage repair and maintenance of genomic integrity. Germ-line mutation Gln 564 His discovered in linker region of BARD1 leads to loss of binding to Cleavage stimulating factor (CstF50), which in turn instigates the premature mRNA transcript formation and apoptosis. We have studied the dynamics of ARD domain present in the BARD1 wild-type and mutant protein in association with CstF50 using biophysical, biochemical and molecular dynamics simulations. It has been observed that the ARD domain is relatively more flexible than the BRCT domain of BARD1. Further relative orientations of both the ARD and BRCT domains varies due to the highly flexible nature of the connecting linker region present between the domains. It has been observed that mutant ARD domain is more dynamic in nature compared to wild-type protein. Molecular docking studies between BARD1 Gln 564 His mutant and CstF50 shows the loss of interactions. Furthermore, domain motion of ARD present in BARD1 was stabilized when complexed with CstF50.

BARD1 (BRCA1 associated RING domain protein 1), as an important animal tumor suppressor gene associated with many kinds of cancers, has been intensively studied for decades. Surprisingly, homolog of BARD1 was found in plants and it was renamed AtROW1 (repressor of Wuschel-1) according to its extremely important function with regard to plant stem cell homeostasis. Although great advances have been made in human BARD1, the function of this animal tumor-suppressor like gene in plant is not well studied and need to be further elucidated. Here, we review and summarize past and present work regarding this protein. Apart from its previously proposed role in DNA repair, recently it is found essential for shoot and root stem cell development and differentiation in plants. The study of AtROW1 in plant may provide an ideal model for further elucidating the functional mechanism of BARD1 in mammals.